Promiscuous HER-2/Neu CD4 T cell epitopes

ABSTRACT

The present invention relates to the discovery of novel T cell epitopes of the human HER-2/Neu protein that is promiscuous for at least 25 different HLA-DR alleles. The invention also relates to compositions that contain one of the novel epitopes or a fusion peptide of such a epitope and a heterologous polypeptide. Further disclosed herein is the use of the epitopes or their fusion peptides, and compositions containing the epitopes or their fusion peptides.

RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.11/836,645, filed Aug. 9, 2007, now U.S. Pat. No. 7,972,602, and U.S.Patent Application Ser. No. 60/837,209, filed Aug. 11, 2006, thecontents of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

HLA class II-restricted CD4+ T cells play a critical role in cellularimmunity and are a key component of anti-tumor immune responses. CD4+ Tcells provide necessary help to tumor-specific CTLs (Topalian 1994. CurrOpin Immunol 6:741-745) and produce cytokines such as interferon gamma(IFNγ), which can activate antigen presenting cells and mediate otherimmunological effects (Corthay et al., 2005. Immunity 22:371-383).Experimental results in several systems have demonstrated that CD4+ Tcells are necessary for an effective anti-tumor immune response. Giventhe importance of CD4+ T cells in generating a robust immune response,an optimally designed cancer immunotherapy or anti-tumor vaccine shouldinduce both tumor-specific CD4+ and CD8+ T cells for maximal efficacy.

The design of cancer immunotherapies and vaccines has benefited greatlyfrom the identification of tumor-associated antigens. One such antigen,HER-2/Neu, is a prime target for such strategies due to itsamplification in a variety of cancers, including breast and ovariancancer. The HER-2/Neu oncogene encodes a transmembrane glycoprotein withhomology to epidermal growth factor receptor (Coussens et al., 1985.Science 230:1132-1139). Overexpression of HER-2/Neu occurs inapproximately 30% percent of breast adenocarcinomas and is associatedwith aggressive disease and a poor prognosis. As a result, severalimmunological approaches designed to increase T cell recognition of theHER-2/Neu protein have been tested in clinical trials. Characterizingthe resulting HER-2/Neu-specific T cell responses in such studies hasled to the identification of several HLA class I and class II-restrictedT cell epitopes within the HER-2/Neu protein (Sotiriadou et al., 2001.Br J Cancer 85:1527-1534). The identification of these epitopes has, inturn, enhanced our ability to detect and quantitate HER-2/Neu-specific Tcell response. Such information leads to improved designs for effectiveimmunotherapies and provides a better understanding of the role ofHER-2/Neu-specific T cell in eradicating HER-2/Neu-expressing tumors.

The usefulness of a defined T cell epitope is limited by itsHLA-restriction. Peptide epitopes typically form productive peptide-MHCcomplexes with a small number of HLA alleles and stimulate T cellresponses only in individuals expressing those alleles. This confinesimmunological studies and clinical trials to individuals of a specificHLA type, often 20% or less of the general population. So-calledpromiscuous T cell epitopes, which can be presented by a larger numberof HLA alleles, have been described for several tumor antigens.Promiscuous T cell epitopes can bind to multiple HLA alleles tostimulate antigen-specific T cells, allowing for the induction and studyof T cell responses in individuals of different HLA types. Additionally,promiscuous epitopes are valuable because the immunotherapies andvaccines based on these epitopes can be widely applicable to the generalpopulation for cancer treatment and prevention. Thus, there exists aclear need for new information relating to previously unknownpromiscuous epitopes of tumor antigens.

The present inventors have identified a series of novel promiscuous Tcell epitopes in the HER-2/Neu protein sequence. These epitopes, locatedwithin the region of 270-284 or 268-286 of the HER-2/Neu protein, arerecognized by a CD4+ T cell clone generated from a patient treated withan autologous, active cellular immunotherapy for HER-2/Neuoverexpressing carcinomas (Valone et al., 2001. Cancer J 7 Suppl2:S53-61). The T cell clone recognizes these peptide epitopes presentedin the context of at least 25 different HLA-DRB1* alleles. Antibodyblocking experiments confirm that the recognition is HLA-DR restricted.Furthermore, these epitopes are naturally processed and presented fromexogenous protein antigen. The promiscuity of these epitopes fordifferent HLA-DRB1* alleles makes these epitopes a valuable tool forevaluating HER-2/Neu-specific immune responses regardless of HLA type.Additionally, these epitopes can be used as a universal CD4 T helpercell epitope in peptide-based vaccines or immunotherapies for thetreatment HER-2/Neu+ cancers.

BRIEF SUMMARY OF THE INVENTION

The present invention describes novel HER-2/Neu epitopes that can bepresented by antigen presenting cells of a number of different HLAalleles to induce a HER-2/Neu specific T cell response. In the firstaspect, this invention provides an isolated peptide derived from asegment of the HER-2/Neu protein sequence (residues 268-286, i.e., SEQID NO:1). The peptide consists of: (a) 12 to 15 contiguous amino acidsof residues 3-17 of SEQ ID NO:1; or (b) 16 to 18 contiguous amino acidsof SEQ ID NO:1. Also provided is a fusion product comprising theHER-2/Neu derived peptide fused to a heterologous polypeptide. In somecases, the peptide is fused to the heterologous polypeptide by a peptidebond, such that the fusion product is in essence a recombinant fusionprotein.

Preferably, the isolated peptide derived from the HER-2/Neu sequence orits fusion product is capable of inducing a T cell immune responsespecific to a HER-2/Neu protein when presented by an antigen-presentingcell of at least 10 different HLA-DR alleles, and more preferably, atleast 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or moredifferent HLA-DR alleles.

In some embodiments, the HLA-DR alleles are selected from the groupconsisting of 0101, 0102, 0103, 1503, 160201, 0301, 0302, 0401, 0402,040301, 040501, 1101, 1102, 1103, 1104, 110401, 1201, 1301, 1302, 1401,1402, 0701, 080101, 080201, and 0901.

In some embodiments, the HER-2/Neu derived peptide has the amino acidsequence of SEQ ID NO:2. In other embodiments, the heterologouspolypeptide is a granulocyte-macrophage colony-stimulating factor(GM-CSF).

In a second aspect, the present invention provides an isolated nucleicacid comprising a polynucleotide sequence encoding a HER-2/Neu derivedpeptide described above or a fusion protein joining a HER-2/Neu derivedpeptide and a heterologous polypeptide by a peptide bond, an expressioncassette comprising the nucleic acid, and a host cell comprising theexpression cassette.

In some cases, the polynucleotide sequence encodes the peptide havingthe amino acid sequence of SEQ ID NO:2. In other cases, thepolynucleotide sequence encodes a fusion protein in which theheterologous polypeptide is GM-CSF.

In some embodiments, the expression cassette is a recombinant viralvector. In other embodiments, the expression cassette directs theexpression of the peptide having the amino acid sequence of SEQ ID NO:2or a recombinant fusion protein in which the heterologous polypeptide isGM-CSF.

In a third aspect, the present invention provides a compositioncomprising a HER-2/Neu derived peptide as described above or a fusionproduct of the peptide fused with a heterologous polypeptide, inaddition to a physiologically acceptable excipient.

In some embodiments, the peptide has the amino acid sequence of SEQ IDNO:2. In other embodiments, the heterologous polypeptide is agranulocyte-macrophage colony-stimulating factor (GM-CSF). In yet otherembodiments, the composition further comprises an antigen-presentingcell, which has the HER-2/Neu derived peptide forming a complex with amajor histocompatibility complex (MHC) molecule on the surface of thecell.

In a fourth aspect, the present invention provides a method for inducingin a patient a T cell immune response specific to a HER-2/Neu protein.This method comprises the step of administering to the patient aneffective amount of the composition comprising a HER-2/Neu derivedpeptide as described above or a fusion product of the peptide fused witha heterologous polypeptide, as well as a physiologically acceptableexcipient.

In some embodiments, the peptide has the amino acid sequence of SEQ IDNO:2. In other embodiments, the heterologous polypeptide is agranulocyte-macrophage colony-stimulating factor (GM-CSF).

In a fifth aspect, the present invention provides a method for detectingin a patient a T cell immune response specific to a HER-2/Neu protein.This method comprises the following steps: (a) obtaining anantigen-presenting cell and a T cell from the patient; (b) contactingthe antigen-presenting cell and the T cell with a HER-2/Neu derivedpeptide or a fusion product comprising the peptide and a heterologouspolypeptide; and (c) detecting a T cell response, wherein the detectionof a T cell response indicates the presence of a T cell immune responsespecific to a HER-2/Neu protein in the patient.

In some embodiments, step (c) is performed by ELISPOT, proliferationassay, or flow cytometry. In other embodiments, the HER-2/Neu derivedpeptide has the amino acid sequence of SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. T cell clone HER500.23c21 is HER-2/Neu specific. (A) HER500(amino acids ▪ and BA7072 (HER500/GM-CSF fusion protein) ♦ specific IFNγproduction by HER500.23c21 was measured using ELISA after autologousPBMC presentation of the antigens; background was also tested ▴. Assaywas set up in triplicate in 96 well round bottom plates in complete IMDMmedia with 10% FBS. Autologous PBMCs were used at 2×10⁵ cells/well and Tcell clone HER500.23c21 was added at 1×10⁵ cells/well. Antigens were atthe final concentrations shown. Assay was incubated for 48 hours at 37°C. with 5% CO₂ and supernatant was removed from wells to test forcytokine production. (A) IFNγ production. Data points shown representthe calculated pg/mL for each antigen concentration. (B) IL-2 productionfor the same assay was measured using HT-2 cell proliferation. Datapoints represent the mean CPM values for each antigen concentration.

FIG. 2. T cell clone HER500.23.21 is specific for Peptide #63(HERp270-284). The HER-2/neu epitope was mapped for clone HER500.23.21and specificity was determined using autologous EBV-LcLs and eachindividual HER500 peptide #1-125 (15 amino acids in length). The assaywas set up in 96 well round bottom plates with autologous EBV-LcLs at2×10⁵ cells/well and the T cell clone HER500.23.21 added at 1×10⁵cells/well. Each peptide was used at a final concentration of 1 ug/mL.The assay was incubated at 37° C. with 5% CO₂ for 48 hours at which timesupernatant was removed to test for cytokine production. (A) IFNγproduction by clone HER500.23.21 was measured using ELISA, calculatedpg/mL is shown for each HER500 peptide. (B) IL-2 production was measuredusing HT-2 cell proliferation.

FIG. 3. HLA-DR Restriction. Autologous EBV-LcL at 2×10⁵ cells/mL andHERp270-284 was used to stimulate the T cell clone HER500.23c21 (1×10⁵cells/well), with and without the addition of blocking anti-MHC class IIantibodies. Anti-HLA-DR ♦ and anti-HLA-DQ/anti-HLA-DP ▪ were titrated atthe concentrations shown in cRPMI+10% FBS. Wells also containedEBV-LcLs, HER500.23c21T cell clone and peptide (1 μg/mL). Assay wasincubated for 48 hours at 37° C. with 5% CO₂. Supernatants wereharvested and frozen at −20° C. and at a later date, analyzed for IFNγproduction using ELISA. Multiple experiments were performed, meantriplicate values for one representative experiment is shown.

FIG. 4. HERp270-284 is a promiscuous MHC Class II HER-2/Neu epitope. (A)EBV-LcL lines homozygous for various HLA-DR alleles were tested fortheir ability to present HERp270-284 to clone HER500.23c21.Antigen-specific IFNγ production by HER500.23c21 was above the upperdetection limit of the IFNγ ELISA (4000 pg/mL) with all EBV-LcL lines.Negative controls included an irrelevant HER500 peptide, no peptide andeach EBV-LcL line alone (all values not detectable, not shown). Theassay was set up in 96 well round bottom plates in cRPMI+10% FBS with2×10⁵ EBV-LcL/well and 1×10⁵ HER500.23c21 cells/well. Peptides were usedat a final concentration of 1 μg/mL. The assay was incubated for 48hours at 37° C. with 5% CO₂. Supernatants were harvested, frozen at −20°C. and then analyzed for IFNγ by ELISA. Results are shown for onerepresentative experiment. (B) HERp270-284 was titrated with each EBVLcL line to determine the sensitivity of HER500.23c21 to differentalleles. The assay was set up as in (A) except HERp270-284concentrations are: 10 ng/mL, 33 ng/mL, 66 ng/mL, 100 ng/mL and 250ng/mL. All HERp270-284 concentrations above 250 ng/mL resulted in IFNγproduction by HER500.23c21 above the upper detection limit of the IFNγELISA (4000 pg/mL). Results are shown for one representative experiment.

FIG. 5. Graphic depiction of IFNγ production by HER500.23c21 followingexposure to peptides presented by EBV LcL lines of diverse HLA-DRb1alleles. The peptides were 9-mers (SEQ ID NOS:3-9) within the HER-2/Neupeptide 270-284 (SEQ ID NO:2).

FIG. 6. Graphic depiction of IFNγ production by HER500.23c21 followingexposure to peptides presented by EBV LcL lines of diverse HLA-DRb1alleles. The peptides were 10- or 11-mers (SEQ ID NOs:12-15, 10, 1,16-19 and 11, respectively) within the HER-2/Neu peptide 270-284.

FIG. 7. Graphic depiction of IFNγ production by HER500.23c21 (measuredby absorbance at 492 nm) following exposure to peptides presented by EBVLcL lines of diverse HLA-DRb1 alleles. The peptides were 12-mers (SEQ IDNOs:20-23) within the HER-2/Neu peptide 270-284.

FIG. 8. Graphic depiction of IFNγ production by HER500.23c21 (indicatedby absorbance at 492 nm) following exposure to peptides presented by EBVLcL lines of diverse HLA-DRb1 alleles. The peptides were 13-mers (SEQ IDNOs:24-26) within the HER-2/Neu peptide 270-284.

FIG. 9. Graphic depiction of IFNγ production by HER500.23c21 (indicatedby absorbance at 492 nm) following exposure to peptides presented by EBVLcL lines of diverse HLA-DRb1 alleles. The peptides were 14-mers (SEQ IDNOs:27 and 28) within the HER-2/Neu peptide 270-284.

FIG. 10. Graphic depiction of IFNγ production by HER500.23c21 (indicatedby absorbance at 492 nm) following exposure to peptides presented by EBVLcL lines of diverse HLA-DRb1 alleles. The peptides were 16-mers (SEQ IDNOs:29 and 30) within the HER-2/Neu peptide 268-286.

FIG. 11. Graphic depiction of IFNγ production by HER500.23c21 (indicatedby absorbance at 492 nm) following exposure to peptides presented by EBVLcL lines of diverse HLA-DRb1 alleles. The peptides were 17-mers (SEQ IDNOs:31-33) within the HER-2/Neu peptide 268-286.

DEFINITIONS

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

In this application, the term “amino acid” refers to naturally occurringand synthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure different from the generalchemical structure of an amino acid, but capable of functioning in amanner similar to a naturally occurring amino acid.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol.Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, or mRNA encoded by a gene.

When the relative locations of elements in a polynucleotide sequence areconcerned, a “downstream” location is one at the 3′ side of a referencepoint, and an “upstream” location is one at the 5′ side of a referencepoint.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds. In this application, the aminoacid sequence of a polypeptide is presented from the N-terminus to theC-terminus. In other words, when describing an amino acid sequence of apeptide, the first amino acid from the N-terminus is referred to as the“first amino acid.”

When used in the context of describing partners of a fusion peptide, theterm “heterologous” refers to the relationship of one peptide fusionpartner to the another peptide fusion partner: the manner in which thefusion partners are present in the fusion peptide is not one that can befound a naturally occurring protein. For instance, a “heterologouspolypeptide” fused with a HER-2/Neu epitope to form a fusion peptide maybe one that is originated from a protein other than a HER-2/Neu protein,such as a granulocyte-macrophage colony-stimulating factor (GM-CSF). Onthe other hand, a “heterologous polypeptide” may be one derived fromanother portion of the HER-2/Neu protein that is not immediatelycontiguous to the HER-2/Neu epitope. A “heterologous polypeptide” maycontain modifications of a naturally occurring protein sequence or aportion thereof, such as deletions, additions, or substitutions of oneor more amino acid residues. Regardless of the origin of the“heterologous polypeptide” (i.e., whether it is derived from theHER-2/Neu protein or another protein), the fusion peptide should notcontain a subsequence of the human HER-2/Neu that encompasses the aminoacid sequence of SEQ ID NO:1 and have more than 18 amino acids inlength. In some exemplary embodiments, a “heterologous polypeptide” foruse in the present invention has no more than 15-20 amino acids inlength; in other embodiments, a “heterologous polypeptide” has at least100 amino acids in length.

The word “fuse” or “fused,” as used in the context of describing apeptide of this invention that comprises a HER-2/Neu epitope joined witha heterologous polypeptide, refers to a connection between the epitopeand the heterologous polypeptide by any covalent bond, including apeptide bond.

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidwhich contains sequence information for a structural RNA such as rRNA, atRNA, or the primary amino acid sequence of a specific protein orpeptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences that may be introduced to conform to codon preference in aspecific host cell.

An “expression cassette” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular polynucleotidesequence in a host cell. An expression cassette may be part of aplasmid, viral genome, or nucleic acid fragment. Typically, anexpression cassette includes a polynucleotide to be transcribed,operably linked to a promoter.

The term “recombinant,” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of a nucleicacid or protein from an outside source or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,under-expressed or not expressed at all.

The term “administration” or “administering” refers to various methodsof contacting a substance with a mammal, especially a human. Modes ofadministration may include, but are not limited to, methods that involvecontacting the substance intravenously, intraperitoneally, intranasally,transdermally, topically, subcutaneously, parentally, intramuscularly,orally, or systemically, and via injection, ingestion, inhalation,implantation, or adsorption by any other means. One exemplary means ofadministration of a HER-2/Neu peptide of this invention or a fusionpeptide comprising a HER-2/Neu peptide and a heterologous polypeptide isvia intravenous delivery, where the peptide or fusion peptide can beformulated as a pharmaceutical composition in the form suitable forintravenous injection, such as an aqueous solution, a suspension, or anemulsion, etc. Other means for delivering a HER-2/Neu peptide or afusion peptide of this invention includes intradermal injection,subcutaneous injection, intramuscular injection, or transdermalapplication as with a patch.

An “effective amount” of a certain substance refers to an amount of thesubstance that is sufficient to effectuate a desired result. Forinstance, an effective amount of a composition comprising a peptide ofthis invention that is intended to induce an anti-Her-2/Neu immunity isan amount sufficient to achieve the goal of inducing the immunity whenadministered to a subject. The effect to be achieved may include theprevention, correction, or inhibition of progression of the symptoms ofa disease/condition and related complications to any detectable extent.The exact quantity of an “effective amount” will depend on the purposeof the administration, and can be ascertainable by one skilled in theart using known techniques (see, e.g., Lieberman, Pharmaceutical DosageForms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999)).

A “physiologically acceptable excipient” is an inert ingredient used inthe formulation of a composition of this invention, which contains theactive ingredient(s) of a HER-2/Neu peptide or a fusion peptidecomprising a HER-2/Neu peptide and a heterologous polypeptide and issuitable for use, e.g., by injection into a patient in need thereof.This inert ingredient may be a substance that, when included in acomposition of this invention, provides a desired pH, consistency,color, smell, or flavor of the composition.

As used herein, the term “T cell immune response” refers to activationof antigen specific T cells as measured by proliferation or expressionof molecules on the cell surface or secretion of proteins such ascytokines.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present inventors have identified a series of novel promiscuous Tcell epitopes from the HER-2/Neu protein. These peptide epitopesdemonstrate remarkable HLA promiscuity as they can be presented in thecontext of at least 25 different HLA-DRB1 alleles. The presentation ofthese epitopes by such a wide range of HLA-DRB1 alleles makes theseepitopes extremely valuable as universal CD4 T helper cell epitopes inpreparation of vaccines or immunotherapies for the treatment of cancersoverexpressing HER-2/Neu in the general human population.

II. Chemical Synthesis of Peptides

The peptides of the present invention, particular those of relativelyshort length (e.g., no more than 50-100 amino acids), may be synthesizedchemically using conventional peptide synthesis or other protocols wellknown in the art.

Peptides may be synthesized by solid-phase peptide synthesis methodsusing procedures similar to those described by Merrifield et al., J. Am.Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-PhasePeptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Grossand Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980);and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem.Co., Rockford, Ill. (1984). During synthesis, N-α-protected amino acidshaving protected side chains are added stepwise to a growing polypeptidechain linked by its C-terminal and to a solid support, i.e., polystyrenebeads. The peptides are synthesized by linking an amino group of anN-α-deprotected amino acid to an α-carboxy group of an N-α-protectedamino acid that has been activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc, which is acid labile, and Fmoc,which is base labile.

Materials suitable for use as the solid support are well known to thoseof skill in the art and include, but are not limited to, the following:halomethyl resins, such as chloromethyl resin or bromomethyl resin;hydroxymethyl resins; phenol resins, such as4-(α-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin;tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins arecommercially available and their methods of preparation are known bythose of ordinary skill in the art.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe solid support. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arethen cleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein (See, e.g., Athertonet al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press(1989), and Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed.,Springer-Verlag (1993)).

III. Recombinant Production of Peptides

A. General Recombinant Technology

Basic texts disclosing general methods and techniques in the field ofrecombinant genetics include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Ausubel et al., eds.,Current Protocols in Molecular Biology (1994).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett.22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purificationof oligonucleotides is performed using any art-recognized strategy,e.g., native acrylamide gel electrophoresis or anion-exchange HPLC asdescribed in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

Recombinant production is an effective means to obtain peptides of thisinvention, particularly those of relatively large molecular weight, forexample, a fusion peptide of a HER-2/Neu epitope and a GM-CSF. Thesequence of a polynucleotide encoding a peptide of this invention, andsynthetic oligonucleotides can be verified after cloning or subcloningusing, e.g., the chain termination method for sequencing double-strandedtemplates of Wallace et al., Gene 16: 21-26 (1981).

B. Construction of an Expression Cassette

Obtaining a Polynucleotide Sequence Encoding a Peptide of the Invention

A polynucleotide sequence encoding a peptide of this invention can beobtained by chemical synthesis, or can be purchased from a commercialsupplier, which may then be further manipulated using standardtechniques of molecular cloning.

Modification of Nucleic Acids for Preferred Codon Usage in a HostOrganism

The polynucleotide sequence encoding a peptide of this invention can beoptionally altered to coincide with the preferred codon usage of aparticular host. For example, the preferred codon usage of one strain ofbacterial cells can be used to derive a polynucleotide that encodes apeptide of the invention and includes the codons favored by this strain.The frequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell (e.g., calculation service isavailable from web site of the Kazusa DNA Research Institute, Japan).This analysis is preferably limited to genes that are highly expressedby the host cell.

At the completion of modification, the coding sequences are verified bysequencing and are then subcloned into an appropriate expression vectorfor recombinant production of the peptides of this invention.

Following verification of the coding sequence, the peptide of thepresent invention can be produced using routine techniques in the fieldof recombinant genetics.

C. Expression Systems

To obtain high level expression of a nucleic acid encoding a peptide ofthe present invention, one typically subclones a polynucleotide encodingthe peptide into an expression vector that contains a strong promoter todirect transcription, a transcription/translation terminator and aribosome binding site for translational initiation. Suitable bacterialpromoters are well known in the art and described, e.g., in Sambrook andRussell, supra, and Ausubel et al., supra. Bacterial expression systemsfor expressing a peptide of this invention are available in, e.g., E.coli, Bacillus sp., Salmonella, and Caulobacter. Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known inthe art and are also commercially available. In one embodiment, theeukaryotic expression vector is an adenoviral vector, anadeno-associated vector, or a retroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically includes atranscription unit or expression cassette that contains all theadditional elements required for the expression of a peptide of thisinvention in host cells. A typical expression cassette thus contains apromoter operably linked to the polynucleotide sequence encoding thepeptide and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding the peptide is typically linked to acleavable signal peptide sequence to promote secretion of the peptide bythe transformed cell. Such signal peptides include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene (e.g., encoding theheterologous polypeptide), introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as abaculovirus vector in insect cells, with a polynucleotide sequenceencoding the peptide of this invention under the direction of thepolyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary. Similar toantibiotic resistance selection markers, metabolic selection markersbased on known metabolic pathways may also be used as a means forselecting transformed host cells.

When periplasmic expression of a recombinant protein (e.g., a peptide ofthe present invention) is desired, the expression vector furthercomprises a sequence encoding a secretion signal, such as the E. coliOppA (Periplasmic Oligopeptide Binding Protein) secretion signal or amodified version thereof, which is directly connected to 5′ of thecoding sequence of the protein to be expressed. This signal sequencedirects the recombinant protein produced in cytoplasm through the cellmembrane into the periplasmic space. The expression vector may furthercomprise a coding sequence for signal peptidase 1, which is capable ofenzymatically cleaving the signal sequence when the recombinant proteinis entering the periplasmic space. More detailed description forperiplasmic production of a recombinant protein can be found in, e.g.,Gray et al., Gene 39: 247-254 (1985), U.S. Pat. Nos. 6,160,089 and6,436,674.

C. Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian,yeast, insect, or plant cell lines that express large quantities of apeptide of this invention, which are then purified using standardtechniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622(1989); Guide to Protein Purification, in Methods in Enzymology, vol.182 (Deutscher, ed., 1990)). Transformation of eukaryotic andprokaryotic cells are performed according to standard techniques (see,e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss,Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA, or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe peptide of this invention.

D. Detection of Recombinant Expression of a Peptide in Host Cells

After the expression vector is introduced into appropriate host cells,the transfected cells are cultured under conditions favoring expressionof the peptide of this invention. The cells are then screened for theexpression of the recombinant peptide, which is subsequently recoveredfrom the culture using standard techniques (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well knownamong those skilled in the art. First, gene expression can be detectedat the nucleic acid level. A variety of methods of specific DNA and RNAmeasurement using nucleic acid hybridization techniques are commonlyused (e.g., Sambrook and Russell, supra). Some methods involve anelectrophoretic separation (e.g., Southern blot for detecting DNA andNorthern blot for detecting RNA), but detection of DNA or RNA can becarried out without electrophoresis as well (such as by dot blot). Thepresence of nucleic acid encoding a peptide of this invention intransfected cells can also be detected by PCR or RT-PCR usingsequence-specific primers.

Second, gene expression can be detected at the polypeptide level.Various immunological assays are routinely used by those skilled in theart to measure the level of a gene product, particularly usingpolyclonal or monoclonal antibodies that react specifically with apeptide of the present invention, particularly one containing asufficiently large heterolougs polypeptide (e.g., Harlow and Lane,Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988;Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniquesrequire antibody preparation by selecting antibodies with highspecificity against the peptide or an antigenic portion thereof. Themethods of raising polyclonal and monoclonal antibodies are wellestablished and their descriptions can be found in the literature, see,e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976).

IV. Purification of Peptides

A. Purification of Chemically Synthesized Peptides

Purification of synthetic peptides is accomplished using various methodsof chromatography, such as reverse phase HPLC, gel permeation, ionexchange, size exclusion, affinity, partition, or countercurrentdistribution. The choices of appropriate matrices and buffers are wellknown in the art.

B. Purification of Recombinantly Produced Peptides

1. Purification of Peptides from Bacterial Inclusion Bodies

When a peptide of the present invention is produced recombinantly bytransformed bacteria in large amounts, typically after promoterinduction, although expression can be constitutive, the peptides mayform insoluble aggregates. There are several protocols that are suitablefor purification of protein inclusion bodies. For example, purificationof aggregate proteins (hereinafter referred to as inclusion bodies)typically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, anon-ionic detergent. The cell suspension can be ground using a Polytrongrinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the cellscan be sonicated on ice. Alternate methods of lysing bacteria aredescribed in Ausubel et al. and Sambrook and Russell, both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, may be inappropriate foruse in this procedure due to the possibility of irreversibledenaturation of the proteins, accompanied by a lack of immunogenicityand/or activity. Although guanidine hydrochloride and similar agents aredenaturants, this denaturation is not irreversible and renaturation mayoccur upon removal (by dialysis, for example) or dilution of thedenaturant, allowing re-formation of the immunologically and/orbiologically active protein of interest. After solubilization, theprotein can be separated from other bacterial proteins by standardseparation techniques. For further description of purifying recombinantpolypeptides from bacterial inclusion body, see, e.g., Patra et al.,Protein Expression and Purification 18:182-190 (2000).

Alternatively, it is possible to purify recombinant polypeptides, e.g.,a peptide of this invention, from bacterial periplasm. Where therecombinant polypeptide is exported into the periplasm of the bacteria,the periplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to those of skill in the art(see e.g., Ausubel et al., supra). To isolate recombinant peptides fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant peptides present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

2. Standard Protein Separation Techniques for Purification

When a recombinant polypeptide, e.g., a peptide of the presentinvention, is expressed in host cells in a soluble form, itspurification can follow the standard protein purification proceduredescribed below. This standard purification procedure is also suitablefor purifying peptides obtained from chemical synthesis.

i. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest, e.g., a peptide of the presentinvention. The preferred salt is ammonium sulfate. Ammonium sulfateprecipitates proteins by effectively reducing the amount of water in theprotein mixture. Proteins then precipitate on the basis of theirsolubility. The more hydrophobic a protein is, the more likely it is toprecipitate at lower ammonium sulfate concentrations. A typical protocolis to add saturated ammonium sulfate to a protein solution so that theresultant ammonium sulfate concentration is between 20-30%. This willprecipitate the most hydrophobic proteins. The precipitate is discarded(unless the protein of interest is hydrophobic) and ammonium sulfate isadded to the supernatant to a concentration known to precipitate theprotein of interest. The precipitate is then solubilized in buffer andthe excess salt removed if necessary, through either dialysis ordiafiltration. Other methods that rely on solubility of proteins, suchas cold ethanol precipitation, are well known to those of skill in theart and can be used to fractionate complex protein mixtures.

ii. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of a protein of interest, e.g., a peptide of the presentinvention. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the peptide of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

iii. Column Chromatography

A protein of interest (such as a peptide of the present invention) canalso be separated from other proteins on the basis of its size, netsurface charge, hydrophobicity, or affinity for ligands. In addition,antibodies raised against a peptide of this invention can be conjugatedto column matrices and the peptide immunopurified. All of these methodsare well known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

C. Confirmation of Peptide Sequence

The amino acid sequence of a peptide of this invention can be confirmedby a number of well established methods. For example, the conventionalmethod of Edman degradation can be used to determine the amino acidsequence of a peptide. Several variations of sequencing methods based onEdman degradation, including microsequencing, and methods based on massspectrometry are also frequently used for this purpose.

D. Modification of Peptides

The peptides of the present invention can be modified to achieve moredesirable properties. The design of chemically modified peptides andpeptide mimics that are resistant to degradation by proteolytic enzymesor have improved solubility or binding ability is well known.

Modified amino acids or chemical derivatives of the HER-2/Neu peptidesor fusion peptides of this invention may contain additional chemicalmoieties of modified amino acids not normally a part of the HER-2/Neuprotein. Covalent modifications of the peptides are within the scope ofthe present invention. Such modifications may be introduced into apeptide by reacting targeted amino acid residues of the peptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The following examples of chemicalderivatives are provided by way of illustration and not by way oflimitation.

The design of peptide mimics which are resistant to degradation byproteolytic enzymes is known to those skilled in the art. See e.g.,Sawyer, Structure-Based Drug Design, P. Verapandia, Ed., N.Y. (1997);U.S. Pat. Nos. 5,552,534 and 5,550,251. Both peptide backbone and sidechain modifications may be used in designing secondary structuremimicry. Possible modifications include substitution of D-amino acids,N^(α)-Me-amino acids, C_(α)-Me-amino acids, and dehydroamino acids. Tothis date, a variety of secondary structure mimetics have been designedand incorporated in peptides or peptidomimetics.

Other modifications include substitution of a natural amino acid with anunnatural hydroxylated amino acid, substitution of the carboxy groups inacidic amino acids with nitrile derivatives, substitution of thehydroxyl groups in basic amino acids with alkyl groups, or substitutionof methionine with methionine sulfoxide. In addition, an amino acid of aHER-2/Neu peptide or a fusion peptide of this invention can be replacedby the same amino acid but of the opposite chirality, i.e., anaturally-occurring L-amino acid may be replaced by its D-configuration.

V. Fusing a HER-2/NEU Epitope with a Heterologous Polypeptide

In one aspect of this invention, a peptide corresponding to a HER-2/Neupromiscuous T cell epitope is attached to a heterologous polypeptide viaa covalent bond to form a fusion peptide, such that the ability of theHER-2/Neu epitope to induce a T cell response is enhanced. Frequently,this covalent bond is a peptide bond and the HER-2/Neu epitope and theheterologous polypeptide form a new polypeptide. This peptide bond maybe a direct peptide bond between the HER-2/Neu epitope and theheterologous polypeptide, or it may be an indirect peptide bond providedby way of a peptide linker between the HER-2/Neu epitope and theheterologous polypeptide.

Other covalent bonds are also suitable for the purpose of fusing theHER-2/Neu peptide with the heterologous polypeptide. For instance, afunctional group (such as a non-terminal amine group, a non-terminalcarboxylic acid group, a hydroxyl group, and a sulfhydryl group) of onepeptide may easily react with a functional group of the other peptideand establish a covalent bond, other than a peptide bond, thatconjugates the two peptides. A covalent connection between a peptide ofa HER-2/Neu epitope and a heterologous polypeptide can also be providedby way of a linker molecule with suitable functional group(s). Such alinker molecule can be a peptide linker or a non-peptide linker. Alinker may be derivatized to expose or to attach additional reactivefunctional groups prior to conjugation. The derivatization may involveattachment of any of a number of molecules such as those available fromPierce Chemical Company, Rockford, Ill.

VI. Functional Assays

A HER-2/Neu epitope of this invention (or a fusion peptide comprising aHER-2/Neu peptide and a heterologous polypeptide) is useful for itscapability to induce a T cell immune response specific to a HER-2/Neuprotein, when the epitope is presented by an antigen-presenting cellthat may have one of at least 10 different HLA-DR alleles, morepreferably at least 15, 20, or 25 different HLA-DR alleles. Variousfunctional assays can be used to confirm the ability of a HER-2/Neuepitope to induce such a HER-2/Neu specific T cell immune response in apromiscuous manner with regard to antigen presenting cells of differentHLA-DR alleles, including proliferation assay and flow cytometry assaysdetecting the binding between a T cell receptor and a peptide epitope orthe production of cytokines by T cells.

The functional assay system used in the Examples of this application isparticularly suitable for this purpose. Briefly, a panel of at least 10,preferably at least 15, 20 or 25, antigen presenting cell lines, eachhomozygous for a different HLA-DR allele, is employed to presentHER-2/Neu-derived peptides to a clone of CD4⁺ T cells (e.g., cloneHER500.23c21) that is specifically responsive to HER-2/Neu protein(e.g., by production of cytokines such as IFNγ or IL-2). Epitope 270-284having the amino acid sequence of SEQ ID NO:2 is used as a positivecontrol, whereas an irrelevant HER-2/Neu derived peptide, no peptide,and each antigen presenting cell line alone are used as negativecontrols for the assays. The assays are set up in multi-welled cellculture plates in appropriate medium with antigen presenting cells andCD4⁺ T cells in each well. Peptides are diluted to a suitableconcentration and added to each well. Following incubation of anappropriate time period, supernatants are collected from the wells andanalyzed for cytokine production, which can be measured by ELISA basedon absorbance at 492 nm. Typically, the effect of a HER-2/Neu class IIpromiscuous eptiope of this invention in inducing a HER-2/Neu specificCD4⁺ T cell response is at least 25% of the effect of HER-2/Neu epitope270-284 (which has the amino acid sequence set forth in SEQ ID NO:2)under the same assay conditions, e.g., at the same molar concentrationand presented by antigen-presenting cells of the same, individual HLA-DRallele. More preferably such effect is at least 30%, 40%, 50%, 60%, 70%,80% or higher of that shown by HER-2/Neu epitope 270-284 under the sameconditions.

VII. Compositions and Administration

The present invention also provides compositions comprising an effectiveamount of (1) a HER-2/Neu peptide; or (2) a fusion peptide comprising aHER-2/Neu peptide and a heterologous polypeptide; or (3) an antigenpresenting cell (APC) with the peptide of (1) or (2) forming a complexwith an MHC molecule on the cell surface for inducing a T cell immuneresponse specific against a HER-2/Neu protein in both prophylactic andtherapeutic applications. Pharmaceutical compositions of the inventionare suitable for use in a variety of drug delivery systems. Suitableformulations for use in the present invention are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). For a brief review of methods for drug delivery, see,Langer, Science 249: 1527-1533 (1990).

Antigen presenting cells (APCs) can be generated for peptide-loading bya variety of methods. The starting raw material is peripheral blood or aleukapheresis with or without mobilization. APCs can be isolated bymultiple methods, e.g., boyant density centrifugation, elutriation,magnetic beads and plastic adherence used alone or in combination. Afterisolation, APCs are cultured for 1-14 days with or without the presenceof cytokines, growth factors, activation agents, and maturation agents.APCs are loaded with a peptide by addition of peptide to the culture inconcentrations from 1 μg to 1 mg/mL for 6-48 hrs. APCs are harvested,washed, and resuspended in a suitable formulation for infusion. APCs canbe delivered fresh or can be kept in frozen storage for delivery at alater time.

The pharmaceutical compositions of the present invention can beadministered by various routes, e.g., subcutaneous, intradermal,transdermal, intramuscular, intravenous, or intraperitoneal. Thepreferred routes of administering the pharmaceutical compositions aresubcutaneous or intradermal at biweekly doses of about 1 μg-10 mg,preferably 50 μg-1 mg, of a peptide of this invention for a 70 kg adulthuman. The appropriate dose may be administered in weekly, biweekly, ormonthly intervals.

Peptide pulsed APCs can be administered by various routes, e.g.,subcutaneous, intradermal, intravenous or intraperitoneal. The peptidepulsed APCs are delivered in weekly, biweekly, or monthly intervals atdoses of 1 million to 10 billion cells.

For preparing pharmaceutical compositions containing a peptide of thepresent invention, inert and pharmaceutically acceptable excipients orcarriers are used. Liquid pharmaceutical compositions include, forexample, solutions, suspensions, and emulsions suitable for intradermal,subcutaneous, parenteral, or intravenous administration. Sterile watersolutions of the active component (e.g., a HER-2/Neu peptide or fusionpeptide) or sterile solutions of the active component in solventscomprising water, buffered water, saline, PBS, ethanol, or propyleneglycol are examples of liquid compositions suitable for parenteraladministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents, detergents, and the like.

Sterile solutions can be prepared by dissolving the active component(e.g., a HER-2/Neu peptide or fusion peptide) in the desired solventsystem, and then passing the resulting solution through a membranefilter to sterilize it or, alternatively, by dissolving the sterilecompound in a previously sterilized solvent under sterile conditions.The resulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the preparationstypically will be between 3 and 11, more preferably from 5 to 9, andmost preferably from 7 to 8.

The pharmaceutical compositions containing a HER-2/Neu peptide or fusionpeptide can be administered for prophylactic and/or therapeutictreatments. In therapeutic applications, compositions are administeredto a patient already suffering from a condition that may be exacerbatedby the proliferation of tumor cells overexpression the HER-2/Neu proteinin an amount sufficient to prevent, cure, reverse, or at least partiallyslow or arrest the symptoms of the condition and its complications. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend on theseverity of the disease or condition and the weight and general state ofthe patient, but generally range from about 1 μg to about 10 mg of theHER-2/Neu peptide or fusion peptide biweekly for a 70 kg patient, withdosages of from about 50 μg to about 1 mg of the peptide biweekly for a70 kg patient being more commonly used. The appropriate dose may beadministered in weekly, biweekly, or monthly intervals.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of a HER-2/Neu peptide or fusion peptide sufficient toeffectively inhibit HER-2/Neu overexpressing tumor cell proliferation inthe patient for therapeutic purposes.

VIII. Method for Detecting T Cell Response Specific to HER-2/NEU Protein

The present invention further provides a method for detecting whether aT cell immune response specific to a HER-2/Neu protein is present in apatient. This method includes the following steps: first, lymphocytesincluding at least a T cell and an antigen-presenting cell are obtainedfrom a patient. Suitable samples that yield such lymphocytes includeblood, tumor infiltrate, and lymph nodes or lymphatic fluids. Second,the T cell and antigen-presenting cells are exposed to a HER-2/Neupeptide (or a fusion peptide comprising the HER-2/Neu peptide and aheterologous peptide) of this invention under conditions that wouldallow proper presentation of a T cell epitope by the antigen-presentingcell to the T cell. Third, signs of a T cell response is measured invitro by means well known in the art such as ELISPOT, proliferationassay, or flow cytometry. When a T cell response is detected by any ofthese methods, it can be concluded that there exists a T cell immuneresponse specific to a HER-2/Neu protein in the patient.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

Example 1

Materials and Methods

Recombinant Proteins and Synthetic Peptides. BA7072 is a proprietaryrecombinant fusion protein manufactured by Dendreon Corporation(Seattle, Wash.) for use in the investigational vaccine APC8024, for thetreatment of HER-2/neu+ cancers. BA7072 contains protein sequences fromboth the extracellular domain (ECD) and intracellular domain (ICD) ofHER-2/neu. HER500 is a recombinant protein also produced by DendreonCorporation containing sequences from both the ECD and ICD of HER-2/neuand is the HER-2/neu portion of the antigen, BA7072. For defining HER500specific immune responses in vitro, 125 peptides were generated from theHER500 protein sequence. These peptides were 15 amino acids in length,overlapping by 11-mers (Genemed Synthesis, South San Francisco, Calif.).HER500 peptide #63 (HERp270-284, ALVTYNTDTFESMPN; SEQ ID NO:2) of the125 HER500 peptides, corresponds to amino acids 270-284 of the naturalHER-2/neu sequence. In addition to the HER500 15-mer peptides generated,9-, 10-, 11-, 12-, 13-, 14-, 16-, and 17-mer peptides were synthesizedfrom residues 268-286 of the HER-2/Neu sequence. These peptides werederived after NH₂— and COOH— terminal truncations or the addition of 1-2amino acids to HERp270-284 to give the peptides described. All HER500peptides were sequenced and determined to be at >95% purity byanalytical HPLC and mass spectroscopy (Genemed Synthesis).

Subject and Healthy Donor Sample Collection. All subject and healthydonor specimens were collected according to investigator sponsoredprotocols approved by the appropriate Investigational Review Board.After receiving informed consent, whole blood samples were collected byvenipuncture into heparinized vacutainer tubes or syringes and preparedfor transport and/or processing. After receipt of blood samples by ourlaboratory, peripheral blood mononuclear cells (PBMC) were collectedunder sterile conditions by density gradient centrifugation and preparedfor use in specified assays.

In Vitro Generation of HER-2/neu specific T cell clones. PBMC from asubject receiving APC8024, an investigational treatment for HER-2/neupositive cancer, were stimulated in a T-25 tissue culture flask with 10μg/mL of BA7072 overnight in RPMI 1640 with 2 mM L-glutamine, 50 U/mLPenicillin, 50 μg/mL Streptomycin and 20 mM HEPES buffer with 10% HumanAB serum (Gemini BioProducts, Calabasas, Calif.) (cRPMI+10% HS). Thefollowing day IFNγ secreting cells were isolated from the PBMC cultureusing the IFNγ Secretion Assay Cell Enrichment and Detection kit(Miltenyi Biotech, Auburn, Calif.). The IFNγ enriched population wasplated by limiting dilution in 96 well round bottom plates with 10 U/mLrecombinant human IL-2 (Invitrogen). Non-IFNγ secreting cells wereirradiated (3000 rads) and added at 50 μL per well to give a finalvolume in all wells of 150 μL. Plates were incubated for seven days at37° C. with 5% CO₂. EBV-transformed lymphoblastoid cells (EBV-LcL) werealso generated from this subject using autologous PBMC and supernatantfrom the B95-8 cell line (ATCC, Manassas, Va.) for the expansion andtesting of autologous T cell clones. On day 7 of the cloning, IFNγsecreting cells were non-specifically expanded in 96 well plates aspreviously described (Yee et al., 2002. Proc Natl Acad Sci USA99:16168-16173). Briefly, to each well, 100 μL of cRPMI+10% HS mediawith 25 U/mL recombinant human IL-2 and 10 ng/mL anti-human CD3 antibody(BD Pharmingen, San Diego, Calif.) was added with 1×10⁴/well irradiatedautologous EBV-LcLs and 1×10⁵/well irradiated allogeneic PBMC. Plateswere incubated for 14 days at 37° C. and then wells were visuallyinspected for positive growth. Growth positive clones, clone HER.23c21and others, were transferred into 24 well plates and expanded usingrIL-2, anti-CD3 and accessory cells as above. Final volume in each wellwas 2.4 mL and accessory cells were increased in number to give2×10⁶/well irradiated allogeneic PBMC and 1×10⁵/well irradiatedautologous EBV-LcL per well.

Identification and Characterization of HER-2/neu specific T cell clones.Clones were screened for antigen specificity using autologous PBMC andantigen or autologous EBV-LcL and Her500 peptides. Stimulations were setup in 96 well round bottom plates in cRPMI+10% FBS media and incubatedfor 48 hours at 37° C. with 5% CO₂. Additionally, clones were stainedfor CD4 and CD8 surface expression by flow cytometry.

Cytokine Production. To determine cytokine production in the antigenspecific stimulation assays, after 48 hours, 200 μL of supernatant waspulled from cultures and tested for both IL-2 and IFNγ production. IL-2production was measured using the IL-2 dependent line HT-2 (ATCC). HT-2cells were grown in IMDM with 10% FBS and 100 uM NEAA, 1 mM SodiumPyruvate, 2 mM L-glutamine, 50 mM Penicillin, 50 U/mL Streptomycin, 20mM HEPES and 20 uM 2-Mercaptoethanol (cIMDM+10% FBS) and fed twice aweek with 20 ng/mL rIL-2. For the assay, 4 days after HT-2 cells weresplit, cells were washed with IMDM+10% FBS to remove all rIL-2, andcells were added to 96 well round bottom plates at 1×10⁴ cells/well incIMDM+10% FBS. Supernatants from antigen specific stimulation were addedto wells and plates were incubated 24-30 hours at 37° C. The followingday 1 μCi of ³[H] TdR was added for the final 6 hours of the assay andplates were harvested to glass fiber filter mats and DNA incorporationof the radioisotope, or proliferative response, was determined by countsper minute using a liquid scintillation counter (PerkinElmer Life andAnalytical Sciences, Inc. Boston, Mass.). IFNγ production was measuredusing anti-human IFNγ antibody pairs for ELISA (BD Pharmingen, SanDiego, Calif.). Briefly, Immulon 4 plates (Thermo Labsystems/VWR,Brisbane, Calif.) were coated overnight with purified anti-human IFNγantibody (NIB42) at 3 μg/mL. The next day coating antibody was discardedand 4% Bovine Serum Albumin (BSA) (Sigma, St. Louis, Mo.) in PBS(Invitrogen) was added to wells and plates were incubated for 2 hours at37° C. Plates were washed with PBS+0.05% Tween 20 and 100 μL ofsupernatant samples from the antigen specific stimulation was added towells and incubated at room temperature for 1.25-2 hours. Plates werewashed and biotinylated anti-human IFNγ antibody (4S.B3) was diluted in1% BSA in PBS (1 μg/mL) and added to plates for 1 hour at roomtemperature. After washing plates, Strep-Avidin HRP (BD Pharmingen) wasdiluted 1:1000 in PBST and added to wells for 30 minutes at roomtemperature. Finally, plates were washed and Sigma® Fast OPD was addedfor 15 minutes in the dark. 2M HCl was added to stop the reaction andplates were read for absorbance at 492 nm on a spectrophotometer.

HLA-DR Restriction and Promiscuity. To determine HLA-DR restriction ofthe T cell epitope HERp270-284, anti-HLA-DR mAb L243, HLA-DQ mAb 1a3 orHLA-DP mAb B7/21 (20-1.25 μg/mL) were cultured with T cell cloneHER.23c21, peptide HERp270-284 and autologous EBV-LcL in cRPMI+10% FBSmedia. Supernatants were harvested after 48 hours and tested for IL-2and IFNγ production. HERp270-284 was also tested for HLA-DR promiscuityusing EBV-LcL lines purchased from the European Collection of CellCultures originating from the 12^(th) International HistocompatibilityWorkshop (IHW) held in Strasbourg, France. The IHW Lines listed in TableI are homozygous for various HLA-DRβ1 alleles and were propagated inRPMI 1640 with 10% Fetal Bovine Serum, 20 mM HEPES, 2 mM L-glutamine, 50mM Penicillin and 50 U/mL Streptomycin (cRPMI+10% FBS) (Invitrogen,Carlsbad, Calif.). To test for MHC class II promiscuity, HERp270-284 wasadded at 1 μg/mL with each separate EBV-LcL line (2×10⁵ cells/well) andthe T cell clone HER.23c21 (1×10⁵ cells/well) in a 96 well round bottomplate in cRPMI+10% FBS media at 37° C. Supernatants were harvested after48 hours and tested for IFNγ production.

Results

APC8024 is an investigational autologous cell immunotherapy forHer2/neu-expressing breast cancer. To characterize the immune responseinduced by this immunotherapy, we isolated T cell clones from a clinicaltrial subject treated with APC8024. One of the CD4+ T cell clonesgenerated, HER500.23c21, showed a specific response to exogenousHER-2/Neu protein sequences presented by autologous PBMCs (FIG. 1). Inthis experiment, HER500.23c21 was stimulated with increasing doses ofHER500, a recombinant protein containing the intracellular andextracellular domains of HER-2/Neu, and BA7072, which consists of HER500expressed as a fusion protein with hGM-CSF. HER500.23c21 specificallyproduced IFNγ and IL-2 in response to both antigens but no cytokineproduction was observed in the absence of antigen. HER500.23c21responded to each of these proteins in a dose dependent manner,indicating that HER500.23c21 recognizes a HER-2/Neu epitope that isnaturally processed and presented from exogenous protein antigen.

Because of the clear response of HER500.23c21 to HER-2/Neu proteinsequences, the specific epitope recognized by HER500.23c21 was mappedusing peptides. HER500.23c21 was tested against a panel of 125individual overlapping 15-mer peptides covering the HER-2/Neu proteinsequences within HER500. Each peptide was used at 1 ug/ml withautologous EBV LCL cells as antigen presenting cells. In theseexperiments, HER500.23c21 responded strongly to the pool of all 125individual peptides, as measured by either IFNγ or IL2 production (FIGS.2A and B, top bar). In addition, Her500.23.c21 responded to only oneindividual peptide, peptide 63, indicating that this peptide containsthe epitope recognized by HER500.23c21 (FIG. 2). Peptide 63 correspondsto amino acids 270-284 of the HER-2/Neu protein sequence and overlapsthe adjacent peptides by 11 amino acids. Thus, the lack of response topeptide 62 or 64 suggests that the HER500.23c21 T cell epitope iscontained entirely in the HERp270-284 sequence and the amino acidscommon to the flanking peptides do not contain the complete epitope forthis T cell clone. To define the minimal epitope recognized byHER500.23c21, we designed 9-mer, 10-mer or 11-mer peptides with NH₂— andCOOH— terminal truncations of HERp270-284. Stimulation of HER500.23c21with these peptides was successful with only 11-mer sequencesVTYNTDTFESM (SEQ ID NO:10) and TYNTDTFESMP (SEQ ID NO:11) when presentedby EBV LcL lines representing HLA-DRB1 alleles *0301, *0302, *1301 and*1402.

In order to determine which HLA molecules were responsible forpresenting HERp270-284 to HER500.23c21, HLA-blocking antibodies wereused to inhibit presentation and T cell activation. HER500.23c21 wasstimulated with 1 ug/ml HERp270-284 and autologous EBV-LcL in theabsence or presence of increasing concentrations of antibodies specificfor HLA-DR or HLA-DP/-DQ. T cell stimulation was determined by IFNγproduction. The antibody against HLA-DR inhibited the stimulation ofHER500.23c21 by HERp270-284 in a dose-dependent manner whereas theHLA-DP/-DQ blocking antibody had no effect on T cell activation, even athigh concentrations of 20 ug/ml (FIG. 3). These results demonstrate thatHER500.23c21 recognition of HERp270-284 is HLA-DR restricted.

To further define the HLA restriction, we tested the ability ofHERp270-284 to activate HER500.23c21 using a panel of 25 EBV LcL lineshomozygous for different HLA-DRB1* alleles, representing 13 DRserological families (Table I). Every EBV-LcL line tested was able toefficiently stimulate HER500.23c21 in an antigen-specific manner,indicating that the HERp270-284 epitope is promiscuous for at least 25HLA-DRB1* alleles (FIG. 4A). In these experiments, 1 ug/ml ofHERp270-284 peptide induced a maximal T cell response, regardless of theEBV-LCL line used. However, differences in the ability of the variousalleles to stimulate Her500.23c21 were evident at lower concentrationsof peptide (FIG. 4B), with some alleles, such as the DRB1*0401, able tostimulate as low as 10 ng/ml, and other alleles were unable to stimulatebelow 250 ng/ml (DRB1*1103). These differences likely reflect a range ofbinding affinities of HERp270-284 for different DRB1* alleles. However,the high degree of promiscuity observed in these experiments suggeststhat HERp270-284 may contain a universal helper T cell epitope forHER-2/Neu.

Discussion

The study of anti-tumor immune responses is often restricted to a smallnumber of antigens presented by specific HLA types due to reagentlimitations. The identification of promiscuous T cell epitopes can helpalleviate those limitations by permitting the analysis of anti-tumorimmune responses in individuals of diverse HLA types. Because of theirvalue, both as a research tool and potential therapeutic, much efforthas been focused on the identification and characterization ofpromiscuous CD4 and CD8 T cell epitopes. Peptide epitopes with varyingdegrees of promiscuity in their HLA-binding have been identified ininfectious disease antigens-HIV (van der Burg et al., 1999. J Immunol162:152-160), mycobacteria (Valle et al., 2001. Clin Exp Immunol123:226-232), and p. falciparum (Contreras et al., 1998. Infect Immun66:3579-3590), as well as tumor antigens such as NY-ESO (Zarour et al.,2002. Cancer Res 62:213-218), MAGE (Consogno et al., 2003. Blood101:1038-1044), Tert (Schroers et al., 2003. Clin Cancer Res9:4743-4755), and Her2/neu (Kobayashi et al., 2000. Cancer Res60:5228-5236). Computer programs such as TEPITOPE (Bian and Hammer.2004. Methods 34:468-475) use known common binding motifs to predictpromiscuous epitopes based on protein sequence and have identified manypotential new T cell epitopes from a variety of sources. The biologicalrelevance of the epitopes identified in silico is being addressed in anumber of systems (Ruiz et al., 2004. Clin Cancer Res 10:2860-2867; andAl-Attiyah and Mustafa. 2004. Scand J Immunol 59:16-24).

In this study, a novel promiscuous T cell epitope was discovered fromthe tumor associated antigen, HER-2/Neu. This T cell epitope wasidentified with a CD4+ T cell clone isolated from a patient treated withan autologous cell immunotherapy for HER-2/Neu+ cancer. The epitope iscontained within amino acids 270-284 of the HER-2/Neu sequence and isnaturally processed and presented from exogenous protein antigen. Thefact that the T cell clone specific for this epitope was isolated froman individual treated with a HER-2/Neu-specific immunotherapy suggeststhat this epitope may be play a role in vivo as part of a clinicallyrelevant anti-tumor immune response. In addition to being identified ina biologically relevant context, this peptide epitope is interestingbecause it has a very broad HLA-DR promiscuity and it can be presentedto the T cell clone HER500.23c21 by at least 25 different HLA-DRB1*alleles representing 13 serological DR families. Most other peptidesidentified as promiscuous T cell epitopes are presented by just a fewdifferent HLA alleles but we have yet to identify an HLA-DRB1* alleleincapable of presenting HERp270-284 to T cell clone HER500.23c21. Therelative lack of MHC restriction for presentation of this epitope makesit an ideal candidate for a universal HER-2/Neu CD4 T cell epitope.

A major goal of tumor immunology is to develop effective cancerimmunotherapies and vaccines against tumor-associated antigens. Suchtreatments are designed to stimulate an anti-tumor immune response toeradicate the tumor. Focusing on specific tumor associated antigens hasbeen successful in generating anti-tumor immune responses and led to theidentification of specific T cell epitopes within some tumor antigens.While most early work focused solely on generating tumor-specific CD8 Tcell responses, there has been a growing appreciation of the importanceof CD4 T cells in generating an effective anti-tumor immunity. Becauseof this, the study of anti-tumor CD4 T cell responses and identificationof class II-restricted T cell epitopes from tumor associated antigenshas expanded. The utility of such epitopes is greatly increased if suchepitopes can be presented by more than one HLA type. Peptide-basedcancer vaccine strategies are hampered by the HLA restriction of thepeptide epitope within the vaccine. The inclusion of promiscuous T cellepitopes such as the one describe herein broadens the usefulness of suchvaccines within the general population. Inducing an immune responseagainst a tumor antigen by a promiscuous epitope is an efficient way toreach a larger percentage of the general population. Such a promiscuousepitope also provides useful means to analyze the response.

TABLE I HLA-defined EBV-LCL cell lines HLA-DRB1* allele DR serologicalfamily Cell line name 0101 DR1 KAS116 0102 DR1 PMG075 0103 DR103 TER-ND1503 DR15 AMAI 160201 DR16 RML 0301 DR17 VAVY 0302 DR18 RSH 0401 DR4BM14 0402 DR4 YAR 040301 DR4 SSTO 040501 DR4 LKT3 1101 DR11 BM21 1102DR11 BM15 1103 DR11 TISI 1104 DR11 BOB 110401 DR11 FPAF 1201 DR12 BM161301 DR13 OMW 1302 DR13 EMJ 1401 DR14 EK 1402 DR14 AMALA 0701 DR7 BER080101 DR8 BM9 080201 DR8 SPL 0901 DR9 T7526Cell lines were obtained from the ECACC European Collection of CellCultures and are listed in the IMGT/HLA cell directory (websiteebi.ac.uk/imgt/hla/cell query.html).

Example 2

In this series of experiments, additional peptides derived from theHER-2/Neu protein were tested for their potential as promiscuous MHCClass II HER-2/neu epitopes. Epitope HERp270-284 (SEQ ID NO:2) was usedas a positive control in the experiments. A panel of EBV-LcL lines,homozygous for various HLA-DR alleles, was used to present HERp270-284,as well as various lengths of peptides within this 15-amino acidsequence or longer length peptides within the 19-amino acid sequence ofHER-2/Neu 268-286 (SEQ ID NO:1), to clone HER500.23c21. Negativecontrols for the assays included an irrelevant HER500 peptide, nopeptide, and each EBV-LcL line alone. Autologous EBV-LcLs were also runwith each condition as a control for clone HER500.23c21 specificity. Theassays were set up in 96 well round bottom plates in cRPMI+10% FBS with2×10⁵ EBV-LcL/well and 1×10⁵ HER500.23c21 cells/well. Peptides werediluted at a final concentration of 1 μg/mL added at 100 μL/well. Theassay was incubated for 48 hours at 37° C. with 5% CO₂. Supernatantswere harvested and analyzed for IFNγ production using ELISA. Results areshown for one representative experiment for each peptide series (FIGS.5-11). Results of the IFNγ produced by clone HER500.23c21 in response topeptide and each HLA-DR allele are reported in pg/mL of IFNγ or asabsorbance at 492 nm.

All patents, patent applications, and other publications cited in thisapplication, including published amino acid or polynucleotide sequences,are incorporated by reference in the entirety for all purposes.

Sequence Listing

(268-286 of HER-2/Neu protein) SEQ ID NO: 1 CPALVTYNTDTFESMPNPE(270-284 of HER-2/Neu protein, or residues 3-17 of SEQ ID NO: 1)SEQ ID NO: 2 ALVTYNTDTFESMPN

1. An isolated nucleic acid consisting of a polynucleotide sequenceencoding the amino acid sequence of SEQ ID NOs: 2, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or
 31. 2. An isolated nucleic acid comprising apolynucleotide sequence encoding a fusion peptide consisting of theamino acid sequence of SEQ ID NOs: 2, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 31 and a heterologous polypeptide.
 3. The nucleic acid ofclaim 1 or 2, wherein the polynucleotide sequence encodes the amino acidsequence of SEQ ID NO:2.
 4. The nucleic acid of claim 2, wherein theheterologous polypeptide is a granulocyte-macrophage colony-stimulatingfactor (GM-CSF).
 5. An expression cassette comprising the nucleic acidof claim 1 or
 2. 6. The expression cassette of claim 5, which is arecombinant viral vector.
 7. The expression cassette of claim 5, whichdirects the expression of the nucleic acid of claim 1 or
 2. 8. A hostcell comprising the expression cassette of claim
 5. 9. A host cellcomprising the expression cassette of claim
 6. 10. The isolated nucleicacid of claim 1, wherein the polynucleotide sequence encodes the aminoacid sequence of SEQ ID NOs: 20, 21, 22, or
 23. 11. The isolated nucleicacid of claim 1, wherein the polynucleotide sequence encodes the aminoacid sequence of SEQ ID NOs: 24, 25, or
 26. 12. The isolated nucleicacid of claim 1, wherein the polynucleotide sequence encodes the aminoacid sequence of SEQ ID NOs: 27, 28, 29, or 31.